TPX2基因用于宫颈癌诊断与治疗的实验研究

发布时间：2018-04-07 18:18

本文选题：TPX2　切入点：宫颈癌　出处：《吉林大学》2015年博士论文

【摘要】：目的：本课题拟设三个部分进行实验研究。第一部分从细胞水平和组织水平明确TPX2在宫颈不同生理或病理状态下表达的差异性，了解从正常宫颈上皮到CIN再到宫颈浸润癌组织中的TPX2蛋白的表达水平，并结合病例分析，探讨其与宫颈癌的临床分期、病理分化程度和淋巴转移的关系。第二部分探讨小干扰RNA靶向沉默TPX2基因对宫颈鳞癌SiHa细胞的生物学行为影响。第三部分基于RNAi构建稳转染HeLa细胞株，探讨RNAi抑制TPX2蛋白表达对细胞生物学行为的影响和激活p53、p21、Rb细胞信号转导通路的可能性。方法：选取HPV阳性型的人源宫颈鳞癌SiHa细胞株、宫颈腺癌HeLa细胞株和经医学伦理委员会审查同意后且经病理切片诊断证实的97例不同宫颈组织标本作为实验研究材料。第一部分实验采用Western-Blot免疫印迹分析、RT-PCR和免疫组化研究宫颈不同生理或病理状态下TPX2的表达情况。第二部分实验利用RNAi对SiHa细胞中的TPX2基因进行特异性抑制，通过流式细胞术、MTT和侵袭试验检测TPX2抑制后对细胞生物学行为的影响。第三部分构建重组担载TPX2-shRNA的真核载体质粒，并转染HeLa细胞，构建稳转染HeLa细胞株，采用Q-PCR、Western-Blot免疫印迹分析法、MTT、流式细胞术、TUNEL法观察TPX2-shRNA对HeLa细胞的生物学行为的影响以及E6、TPX2、p53、p21、Rb之间的可能相互作用机理，探讨高危型HPV感染后E6、TPX2激活p53、p21、Rb细胞信号和介导细胞信号转导的可能性。结果：第一部分实验结果为TPX2蛋白和TPX2mRNA在CIN、宫颈鳞癌组织、宫颈腺癌组织、宫颈鳞癌细胞SiHa和宫颈腺癌细胞HeLa均有不同程度的表达，但是在正常宫颈上皮组织内，TPX2几乎不表达；随着病情发展，从CINⅠ到CINⅢ的不同宫颈组织的TPX2蛋白表达逐渐增强，宫颈浸润癌临床II期的TPX2表达水平高于I期，淋巴转移阳性的TPX2表达水平高于阴性，病理学低分化的TPX2表达水平高于高分化。第二部分实验结果为TPX2-siRNA转染细胞后，随转染时间的延长，细胞凋亡率逐渐升高；在转染后出现细胞增殖减慢，细胞增殖受到抑制，细胞侵袭能力下降，细胞周期受到调控，主要表现为G1期细胞比例下降，S期和G2期细胞比例升高。第三部分实验成功构建稳定转染重组TPX2-shRNA质粒的HeLa细胞株，对细胞内TPX2的表达起到了特异性抑制，Western-Blot和Q-PCR实验说明TPX2-shRNA在下调TPX2的同时还能够在转录和翻译水平上下调E6，对p53、p21、Rb也具有下调作用，证实TPX2-shRNA具有激活p53、p21、Rb细胞信号的能力，RNAi沉默TPX2后介导细胞信号转导的具体作用机制可能是TPX2被靶向沉默后恢复野生型p53的功能活性，促进细胞凋亡。RNAi沉默TPX2后对细胞周期的调控主要存在两种途经，一方面，p53通过调控其下游靶基因p21的转录和翻译，刺激p21WAF1/CIP1的表达调控细胞周期，p21WAF1/CIP1能够通过引起Rb蛋白磷酸化调控细胞周期；另一方面，从Rb介导的细胞信号转导通路来看，Rb基因通过G1/S期限制点调控细胞周期，Rb蛋白的磷酸化状态决定了细胞增殖能力，非磷酸化Rb可与转录因子E2F结合，Rb蛋白转变为磷酸化后释放具有活性的游离E2F，调控细胞增殖。E6基因通过其产物E6蛋白使具有抑癌作用的非磷酸化Rb蛋白转化为磷酸化Rb蛋白。野生型p53可以同非磷酸化的Rb作用，通过依赖p53诱导的细胞凋亡途经保护Rb的抑癌功能，促使磷酸化Rb蛋白转化为非磷酸化Rb。此外，高危型HPV的E7蛋白可以结合Rb使E2F游离，活化S期转录。RNAi的效果越好，细胞增殖活性受到抑制的程度越明显、细胞凋亡数量越多，停顿于S期和G2/M期的细胞比例越大。结论：在宫颈鳞状细胞癌、CIN、宫颈腺癌组织中均存在TPX2过表达，TPX2在正常宫颈组织中几乎不表达；在宫颈上皮细胞从正常到癌变的发展过程中，TPX2表达含量逐渐增多；宫颈癌的临床分期、病理分级和转移均与TPX2过表达水平密切相关，TPX2过表达水平越高，临床分期越晚、病理分化越差、越容易发生淋巴结转移。RNAi特异性沉默SiHa、HeLa细胞的TPX2后不仅能够调控细胞周期，使细胞周期在S期及G2/M期停顿，而且能够抑制细胞的增殖活性和侵袭能力，TPX2-shRNA能够使细胞发生凋亡。成功构建了稳转染TPX2-shRNA重组质粒的HeLa细胞株，证实了高危型HPV感染后TPX2在转录和翻译水平上均与E6关系密切，TPX2-shRNA能够激活p53、p21、Rb细胞信号通路，介导细胞信号转导途经、调控细胞周期、诱发细胞凋亡；E6、TPX2通过抑制p53、p21、Rb的抗癌功能，使细胞周期检查点功能缺陷，使正常状态的细胞发生癌变。RNAi干扰能够有效抑制TPX2的过表达，保护p53、p21、Rb的正常功能状态，基于RNAi技术抑制TPX2基因的表达可对宫颈癌的发生和进展起抑制作用，，TPX2基因有望成为一种新的宫颈癌早期诊断和基因治疗的候选靶点，为宫颈癌的临床诊断和治疗提供新的思路。
[Abstract]:Objective: This paper proposed three parts. The first part studied the differences from the cell level and tissue level clear expression of TPX2 in cervical under different physiological or pathological condition, understand the expression of water from normal cervical epithelium to CIN to invasive cervical carcinoma TPX2 protein level, and combined with case analysis, to explore the with the clinical stage of cervical cancer, the relationship between the degree of pathological differentiation and lymph node metastasis. The second part discusses the small interfering RNA targeting TPX2 gene on the biological behavior of cervical squamous cell carcinoma SiHa cells. The effects of the third part of the RNAi construction of stable transfected HeLa cells based on the inhibitory effect of RNAi on expression of TPX2 protein on cell biological behavior and activation of p53 p21, the possibility of signal transduction pathway of Rb cells.
Methods: HPV positive type human cervical squamous cell carcinoma SiHa cell line, cervical cancer cell line HeLa and by the medical ethics committee for examination and approval and 97 cases with different cervical tissue biopsy specimens confirmed the diagnosis as the experimental materials. The first part of the experiment using Western-Blot blot analysis, RT-PCR and immunohistochemical study of cervical the physiological or pathological conditions TPX2 expression. The second part of the experiment using RNAi TPX2 gene on SiHa cells was specifically inhibited by flow cytometry. The effect on cell biological behavior and invasion of MTT TPX2 inhibition test. The third part is to construct the eukaryotic recombinant plasmid carrying TPX2-shRNA, and transfected into HeLa to construct stable cells, transfected HeLa cells, using Q-PCR, Western-Blot blot analysis, MTT, flow cytometry and TUNEL method to observe the effect of TPX2-shRNA on HeLa cells The influence of learning behavior and the possible interaction mechanism between E6, TPX2, p53, p21 and Rb are discussed. The possibility of E6, TPX2 activation p53, p21, Rb cell signal and mediating cell signal transduction after high-risk HPV infection is discussed.
Results: in the first part of the experimental results of TPX2 protein and TPX2mRNA in CIN, cervical squamous cell carcinoma, adenocarcinoma of the uterine cervix, cervical squamous cell carcinoma SiHa cells and cervical cancer cell line HeLa in varying degrees, but in normal cervical epithelial tissues, there was almost no expression of TPX2; with the disease development, expression increased gradually from different cervical tissues I CIN to CIN III TPX2 protein, the expression level of invasive carcinoma of clinical stage II TPX2 cervical lymph node metastasis was higher than that in stage I, TPX2 positive expression level was higher than the negative, pathological differentiation of low TPX2 expression level was higher in high differentiation. Some experimental results for second TPX2-siRNA after the transfection, the transfection time, cell apoptosis the rate increased gradually; cell proliferation in transfected cell proliferation inhibition and cell invasion ability decreased, cell cycle regulated, mainly for the G1 phase of the cell ratio decreased, S And the proportion of cells in G2 phase of HeLa cells increased. The third part of the experiment successfully constructed stably transfected with recombinant plasmid TPX2-shRNA, the expression of TPX2 in endothelial cells plays a specific inhibition, Western-Blot and Q-PCR experiments indicated that the TPX2-shRNA in the TPX2 can also cut down E6, at the level of transcription and Translation of p53, p21, Rb with a reduced role, has confirmed that the TPX2-shRNA activation of p53, p21, Rb cell signal, the specific mechanism of RNAi silencing TPX2 mediated signal transduction may be TPX2 by targeting functional recovery after wild-type p53, promote apoptosis of.RNAi cells after silencing of TPX2 in the regulation of cell cycle there are two main way on the one hand, through the regulation of p53, p21 and its downstream target gene transcription and translation, cell cycle regulation stimulates p21WAF1/CIP1 expression, p21WAF1/CIP1 can induce Rb protein phosphorylation in cell cycle Period; on the other hand, from the cell signal transduction pathway mediated by Rb, Rb gene restriction point of cell cycle regulation by G1/S, the phosphorylation status of Rb determines the ability of cell proliferation, non phosphorylated Rb can bind to the transcription factor E2F and Rb protein phosphorylation after release into free E2F with activity the regulation of cell proliferation,.E6 gene has tumor suppressor role of non phosphorylated Rb protein into phosphorylated Rb protein by product E6 protein. Rb effect of wild-type p53 with non phosphorylation, cell apoptosis induced by p53 dependent protection via the tumor suppressor function of Rb, the phosphorylation of Rb protein into non phosphorylation of Rb. in high-risk HPV E7 protein can be combined with Rb to make E2F free, the better effect of activation of S phase.RNAi transcription, cell proliferation activity was inhibited more obviously, the number of apoptosis cells more pauses in S phase and G2/M phase cells The greater the proportion.
Conclusion: in cervical squamous cell carcinoma, CIN, overexpression of TPX2 exists in cervical adenocarcinoma, TPX2 almost no expression in normal cervical tissue; in the process of development of cervical epithelial cells from normal to cancer, the expression of TPX2 was increased; the clinical staging of cervical cancer, pathological grading and metastasis with TPX2 over expression of TPX2 is closely related to the higher level of expression, the later clinical stage, pathological differentiation, the more prone to lymph node metastasis of.RNAi silencing of SiHa, HeLa cells after TPX2 can not only regulate the cell cycle, the cell cycle arrest in S phase and G2/M phase, and can inhibit the cell proliferation activity and the invasion, TPX2-shRNA can induce cell apoptosis. HeLa cells was successfully constructed stably transfected with TPX2-shRNA recombinant plasmid, confirmed the high-risk HPV infection after TPX2 at the level of transcription and translation are closely related with E6, T PX2-shRNA can activate p53, p21, Rb cell signaling pathway, mediated via cellular signal transduction, cell cycle regulation, apoptosis; E6, TPX2 p21, Rb by inhibiting p53, anti-cancer function, cell cycle checkpoint function defects, the cancerous.RNAi interference can effectively inhibit the TPX2 expression, p53 protection p21, normal cell, normal functional status of Rb, inhibit the occurrence and development of RNAi technology to inhibit TPX2 gene expression in cervical cancer based on TPX2 gene, is expected to become a new candidate target for early diagnosis of cervical cancer and gene therapy, to provide new ideas for clinical diagnosis and treatment of cervical cancer.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.33

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